1.1 Field of the Invention
The present invention relates to the genetic manipulation of plants, particularly to methods and compositions for altering the lipid content in plants and seed. Also provided are compositions and methods for the preparation of transgenic plants that over-express a PEAMT polypeptide.
1.2 Description of Related Art
Flowering plants are unusual in how they synthesize choline (Cho) moieties. In leaves and other vegetative tissues, the first and committing step is N-methylation of phosphoethanolamine (P-EA) to give phosphomonomethylethanolamine (P-MME), and the subsequent N-methylations occur at the phosphobase level, the phosphatidyl base level, or both, depending on the species (Datko and Mudd, 1988a; 1988b; Rhodes and Hanson, 1993) (FIG. A). For example, in spinach and sugar beet leaves, essentially all flux through the last two methylations is at the phosphobase level (Hanson and Rhodes, 1983; Summers and Weretilnyk, 1993), whereas in soybean cells it is at the Ptd-base level (Datko and Mudd, 1988a). In contrast, Cho synthesis in the bacterium Rhodobacter sphaeroides, in fingi, and in mammalian liver proceeds solely via the sequential methylation of phosphatidylethanolamine (Ptd-EA) (Vance et al., 1997; Kanipes and Henry, 1997; Arondel et al., 1993). Nerve tissues have a phosphobase methylation route as well as a phosphatidylbase route, but the first methylation appears not to be restricted to the phosphobase level (Mukherjee et al., 1995; Andriamampanry et al., 1991).
The initial methylation of P-EA in plants is catalyzed by S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT), which has been detected in all species tested (Datko and Mudd, 1988b; Summers and Weretilnyk, 1993; Nuccio et al., 1998). PEAMT was recently purified 5,400-fold from spinach leaves, giving a preparation containing several polypeptides (Smith et al., 1999). This preparation catalyzed methylation of P-MME and phosphodimethylethanolamine (P-DME) as well as P-EA, and these activities co-purified in a constant ratio through the three last steps in the procedure (Smith et al., 1999). These data suggest that PEAMT could be trifunctional, but do not rule out a duo or trio of similar N-methyltransferases that act on different phosphobases. Pathways involving one, two or three N-methyltransferases all have precedents. In R. sphaeroides and liver a single Ptd-EA N-methyltransferase mediates all three methylations (Vance et al., 1997; Arondel et al., 1993) whereas in Saccharomyces cerevisiae and Schizosaccharomyces pombe there are two enzymes, one mediating the first methylation of Ptd-EA and another mediating primarily the last two (Kanipes and Henry, 1997). The phosphobase pathway in nerve tissues has three separate N-methyltransferases (Mukherjee et al., 1995).
Certain plants (e.g., spinach, sugar beet) use large amounts of Cho to produce the osmoprotectant glycine betaine (GlyBet) via the pathway Cho→betaine aldehyde→GlyBet (Rhodes and Hanson, 1993). Because GlyBet accumulation contributes to resistance to salinity and drought stress, there has been much interest in engineering GlyBet synthesis in plants that do not naturally produce it (Nuccio et al., 1999). However, when enzymes for Cho oxidation to GlyBet are expressed in such plants (e.g., tobacco, canola), they accumulate little GlyBet, apparently in part because their endogenous Cho supply is inadequate (Nuccio et al., 1998; Huang et al., 1999). This has focused attention on the pathway of Cho biosynthesis and its regulation (Nuccio et al., 1999).
Biochemical and physiological evidence shows that the PEAMT-mediated step is a control point in the biosynthesis of Cho moieties, and that at least two mechanisms are involved. One is feedback inhibition: PEAMT activity in crude Lemna extracts (Mudd and Datko, 1989a; 1989b) and purified spinach preparations (Smith et al, 1999) is inhibited by P-Cho, and 14C tracer data for sugar beet leaf tissue indicate that this occurs in vivo (Hanson and Rhodes, 1983). Another mechanism may be regulation of PEAMT gene expression. The de novo synthesis of Cho in Lemna, soybean and carrot cells is suppressed by exogenous Cho, and this suppression is accompanied by a decrease in extractable PEAMT activity (Mudd and Datko, 1989a; 1989b). Conversely, salinization in spinach, which increases the consumption of Cho in GlyBet synthesis, causes an increase in PEAMT activity (Weretilnyk et al., 1995). Direct evidence that flux through the PEAMT step limits the synthesis of Cho moieties in viva comes from tobacco engineered to convert Cho to GlyBet; supplying MME or DME increases the flux to Cho and GlyBet but supplying EA does not (Nuccio et al., 1998).
1.3 Deficiencies in the Prior Art
A goal of plant breeding has been to modulate the lipid content in plant seeds, and to alter the levels of various plant oils. Despite the importance of PEAMT as the committing step in the biogenesis of Cho moieties, it has not been cloned or unambiguously characterized with respect to the reaction(s) it catalyzes. Nor has any other plant N-methyltransferase participating in Cho biogenesis been cloned.